The migration of leukocytes through the body and the various lymphoid organs is an essential element of the immune system. While circulating in blood or lymphatic vessels, leukocytes are in a resting and low adhesive state. However, when leukocytes are stimulated by signals from the immune system such as exposure to an immune complex or a chemokine gradient, their integrin adhesion receptors become activated. The activation of the integrins is essential for the many functions of leukocytes. Such functions are, for example, binding to antigen-presenting cells, recirculation through lymph nodes and migration out of the vasculature and through the extracellular matrix to sites of inflammation. The integrin activation needs to be tightly regulated as inappropriate leukocyte adhesion leads to significant injury of normal tissues.
Leukocytes express a specific subset of the integrin family, the β2 integrins of which four members are currently known. They have a common β2 chain (CD 18) but different α subunits (αL or CD11a, αM or CD11b, αX or CD11c, and αD or CD11d) (Gahmberg et al., 1997). The α subunits contain a conserved 200-residue A or I domain, which is essential for binding of most ligands. The crystal structures of I domains from the αL and αM subunits indicate the presence of a cation binding site called the metal-dependent adhesion site. Amino acid substitutions in this site abrogate ligand binding (Huang and Springer, 1995; Kamata et al., 1995).
The major ligands of these integrins, the ICAMs, belong to the immunoglobulin superfamily, and five ICAMs with slightly different binding specificities have been described. The expression of ICAM-1 on endothelial cells is subject to stimulation by inflammatory cytokines, which enhances the β2 integrin-mediated adhesion of leukocytes on endothelial cells. In addition to the ICAMs, fibrinogen and the iC3b complement protein are known ligands of the β2 integrins, particularly of αMβ2 (Mac-1).
Because of the importance of the N integrins for leukocyte function, antagonists of them are potential anti-inflammatory agents. Antibodies to the β2 integrins or the ICAMs have a therapeutic effect in animal models of immunological disorders.
Agents targeting the β2 integrins could also be valuable in the development of therapeutic strategies to human leukemias (Calancette et al., 2000). However, only a few small molecule antagonists of the 62 integrins have been described so far (Kalen et al., 1999; Kelly et al., 1999). Lack of such compounds has prevented the detailed examination of the role of each member of the β2 integrin family in leukemia dissemination as well in inflammatory diseases. In particular, it would be desirable to design compounds that distinguish between the inactive and active state of an integrin. Modeling of such small molecule inhibitors has been hampered by the large size of the peptide ligands developed so far. Linear peptides are often without a well-defined structure when free in solution. Among the few β2 integrin ligands discovered is the 22-amino acid long peptide known as P1 which was derived from ICAM-2 (Li et al., 1993). This peptide retains the leukocyte integrin-activating effect that is typical for ICAM-2 (Li et al., 1995; Kotovuori et al, 1999). Complementary-determining regions of anti-β2 integrin antibodies have been another source to obtain ligand peptides, and one of the isolated peptides, 23 amino acids in length, showed similarity to a sequence present on ICAM-1 (Feng et al., 1998).
To develop smaller peptide ligand leads to the β2 interns, the inventors screened random peptide libraries displayed on filamentous phage. The phage display technique has previously yielded selective peptide ligands to the integrin species α5β1 (Koivunen et al., 1994), αvβ3/β5(Koivunen et al., 1995) and αvβ6 (Kraft et al., 1999). Phage library screenings have also confirmed the earlier findings that the tripeptide sequence RGD is a common recognition sequence of a subset of integins (Pierschbacher and Ruoslahti, 1984). In addition, apparent charged analogues of RGD, such as RLD, KGD, and NGR, have been discovered. The leukocyte integrins α4β1, and α4β7 are known to have a specificity for peptides containing another type of tripeptide sequence, LDV (Komoriya et al., 1991).